Fermentation of eburicoic acid



United States Patent cc 3,377,255

Patented Apr. 9, 1968 3,377,255 amounts and dosages based on the activity of the partic- FERMFENTAIION 0F EBURICOI? P ular compound. The compounds of this invention may Allen I. Laskin, Gnggstown, and Josef Fried, Princeton be administered topically alone, or in combination with N..I., assignors, by mesne assignments, to E. R. Squ1bb a pharmaceutically accwtable Garden gg g f New York a corporation of 5 The compounds of this invention may be prepared ac- No Drawing. Original application Sept. 13, 1963, Ser. No. E 9 Processes P thls f' remploylqg a 308,696 now Patent No. 32 99 dated July 4, 1961 triterpenoi d ac1d as a startlng material. Among the triter- Divided and this application Nov. 23, 1966, Ser. No. PeIlOid acids Which y be employed in this invention 608,713 are included such acids as eburiocoic acid, tumulosic 2 Claims- 10 acid, pinicolic acid, polyporenic acid, elemolic acid, elemonic acid, dehydroeburicoic acid, dehydroelemolic acid, dehydroelemonic acid, sulfurenic acid and other like acids. In the case of such acids as tumulosic acid and ABSTRACT OF THE DISCLOSURE Fermentation of eburicoic acid with microorganisms shlfllfenic acid, a 16 and Sllhsthllent is Preseht p of the genus Glomerella to produce oxygenated deriva- 15 tlvely. As the steps of the process are carried out the tives thereof, final products of this invention when derived from tumulosic or sulfurenic acid will possess a 16 or 15 sub- This application is a division of our application Ser. i as the case may sulfpremf: and 1S .denved No 308 696 filed Sept 13 1963 and now Us liatent according to the procedure described in copending ap- 3 20 plication Ser. No. 308,677 filed on even date herewith This invention relates to and has for its objects the m the name of Josef Fned now US PatePt provision of new physiologically active compounds, proci f f be noted h when tumuloslc ac1d S111 esses for their production and new intermediates useful fllrehlc held 18 employed In the Processes Set forth here i preparing th a inafter, the compounds derived are the 16 and 15 sub- The final products of this invention may be represented stituted'derivatives of the compounds obtained in the by the formulae employment of eubricoic acid. These 16 and 15 substi- X fillHz RooC X ROHiC l R I RI! A ROOC Rome HO\/ HO X m ROOC- X ROHzC I CH3 ROOO and ROHHO HO HQ HaC CH3 H3O CH:

wherein Y is selected from the group. consisting of lower tuted derivatives are also new compounds of this invenalkyl (e.g. methyl) and hydroxy lower alkyl (e.g. hytion.

droxy methyl); X is selected from the group consisting [In this application and in the appended claims, whenof hydrogen and hydroxy; each R' is selected from the ever in the formulae set forth herein a curved line (i) group consisting of hydrogen and lower alkyl; each R" is employed in the linkage of atoms, it is meant to deis selected from the group consisting of hydrogen, hynote that the connected atom may be either in the alpha droxy and acyloxy; and R is selected from the group or beta position, as is determined in the respective comconsisting of hydrogen and acyl. pounds involved] The final products of this invention possess antibiotic The steps of the process of this invention may be repactivity and may be effectively employed for such purresented by the following equations employing eburicoic poses against such microorganisms as penicillin resistant acid as the starting material, wherein R, X and Y are strains of Staphylococcus aureus. The final products of as hereinbefore defined, R maybe hydrogen or acyl; and this invention may be employed for such purposes in the R may be hydrogen or lower alkyl.

Eburicoie Acid I II In the first step of the process of this invention, the chosen triterpenoid acid is subjected to the action of a microorganism of the genus Glomerella, or to the action of the enzymes thereof, under oxidizing and preferable aerobic conditions, and further, this new compound may be further treated to yield the other new compounds of this invention.

The microorganisms of the genus Glomerella which may be employed in the practice of this invention include such microorganisms as Glomerella jusarioides, G. glycines and G. cingulata.

To prepare the compounds of this invention, a triterpenoid acid is first subjected to the action of enzymes of a microorganism of the genus Glomerella under oxidizing conditions. This oxidation can best be efiected either by including the triterpenoid acid in an aerobic culture of the microorganism, or by bringing together, in an aqueous medium, the compounds, air and enzymes of non-proliferating cells of the microorganism.

In general, the conditions of culturing the Glomerella microorganism for the purposes of this invention are (except for the inclusion of the triterpenoid acid to be converted), the same as those of culturing various other microorganisms for the production of antibiotics, vitamin B and other like substances, The microorganism is grown aerobically in contact with (in or on), suitable fermentation medium. A suitable medium essentially comprises a source of carbon and energy. The latter may be a carbohydrate (such as sucrose, molasses, glucose, maltose, starch, or dextrin), a fatty acid, a fat and/or the compounds itself. Preferably, however, the medium includes an assimilable source of carbon and energy in addition to the steroid. Among the fats utilizable for the purpose of this invention are: lard oil, soybean oil, linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesame oil, crude palm oil, fancy mutton tallow, sperm oil, olive oil, tristearin, tripalmitin, triolein and trilaurin. Among the fatty acids utilizable for the purpose of this invention are: stearic acid, palmitic acid, oleic acid, linoleic acid and myristic acid.

The source of nitrogenous factors utilizable for the purposes of this invention may be organic (e.g. soybean meal, cornsteep liquor, meat extract and/or distillers solubles) or synthetic (i.e., composed of simple, synthesizable organic or inorganic compounds such as ammonium salts, alkali nitrates, amino acids or urea).

An adequate sterile air supply should be maintained during fermentation, for example, by the conventional methods of exposing a large surface of the medium to air,

or by utilizing submerged aerated culture. The compound may be added to the culture during the incubation period, or included in the medium prior to sterilization or inoculation. The preferred (but not limiting) range of the concentration of the compound in the culture is about 0.01 to about 0.10%. The culture period (or rather the time of subjecting the compound to the action of the enzyme) may vary considerably, the range of about 1 to 7 days being feasible, but not limiting.

The process yields, inter alia, 3,4-seco-A -eburicadiene-4-ol-3,2l-dioic acid (Compounds A, wherein R and R"=H), which may be separated from the broth by extraction or filtration and from other concomitantly produced compounds by fractional crystallization.

The 3fleece-M -eburicadiene-4-ol-3,Zl-dioic acid may then be alkylated as by treatment with an alkylating agent, for example, ethereal diazomethane to obtain the dimethyl ester thereof (Compounds A, wherein R =R'=CH The monomethyl ester of Compounds A may then be obtained by treatment of the dimethyl ester with an alkali metal base, such as potassium hydroxide. Compounds A are new compounds of this invention.

Compounds A may then be reduced as by treatment with a reducing agent, e.g., palladium on charcoal, to yield the 3,4-seco-A -eburicene-4-ol-3,21-dioie acids (Compounds C, wherein Y=CH which are also new products of this invention.

Compounds A may also be treated with an oxidizing agent, e.g., osmium tetroxide, to yield the 3,4-seco-A eburicene-4,24,28-triol-3,2l-dioic acid (Compounds C, wherein Y=CH OH) which also are new compounds of this invention.

The preferred acyl radicals of this invention are those of hydrocarbon carboxylic acids of less than twelve carbon atoms, as exemplified by the lower alkanoic acids (e.g. acetic, propionic, butyric and tert.-pentanoic acids), the lower alkanoic acids, the monocyclic aryl carboxylic acids (e.g., benzoic and toluic acids), the monocyclic aryl lower alkanoic acids (e.g., phenacetic and ,B-phenylpropionic acid), the cycloalkane carboxylic acids and the cycloalkene carboxylic acids.

The invention may be further illustrated by the following examples:

Example 1.3,4seco-A -eburicadiene-4-0l-3,21- dioic acid (I) (A) Fermentation of eburicoic acid A fermentation medium of the following compositions is prepared:

Grams Dextrose 10 Cornsteep liquor 6 NH H PO 3 Difco yeast extract 2.5 CaCl;, 2.5

Water to make 1 liter.

The pH of the medium is adjusted to 70:0.1 with 2 N NaOH solution, and 50 ml. portions of the medium are distributed in 250 ml. Erlenmeyer flasks, the flasks plugged with cotton and sterilized by autoclaving for 30 minutes at 120 C. When cool, two of the flasks are each inoculated with 1 ml. of a suspension of the surface growth of 21-day old agar slant (10 g. glucose; 2.5 g. yeast extract; 1 g. K i-IP 20 g. agar; distilled Water to one liter) culture of Glomerella fusarioides (ATCC 9552), the suspension being made in 2.5 m1. of Water with 0.01% of sodium lauryl sulfate as a Wetting agent.

The flasks are then mechanically shaken for 72 hours at 25 C. on a 280 cycle per minute rotary shaker, after which about 10% v./-v.) is transferred to each of 50 flasks each containing 50 ml. of the fresh sterile fermentation medium described above. E'buricoic acid is then added by suplementing each flask with 0.25 ml. of a sterile solution of the steroid in N,N-dimethylformamide (60 mg./ml.) so that the medium contains 300 ,ug./ml. of the steroid. The flasks are then incubated an additional 168 hours, after which the flasks are harvested and the contents filtered through a Seitz clarifying pad and Washed with successive 50 ml. portions of warm water. The combined filtrate and washings are adjusted to pH 4.0 with glacial acetic acid and the flocculent precipitate which develops is collected by filtration.

(B) Isolation of 3,4-seco-A -eburicadiene-4-ol-3,21- dioic acid KB: max.

Analysis-Cal'ttd. for C H O (502.71): C, 74.06; H, 10.03. Found (after drying at 140 to constant weight): C. 73.61; H, 1059; C, 73.67; H, 1059.

Neutralization equivalent: 250; Calcd. 251 NMR (pyridine): 5.07 (singlet, 2 protons at C 8.50, 8.52

(two methyl groups at C 6 Example 2. 3,4-seco-A -eburicadiene-'4-ol-3,21- dioic acid 3,21-dimethyl ester (II) 3,4-seco-A -eburicadiene-4-ol-3,2l-dioic acid is treated with ethereal diazomethane in six ml. of methanol, to yield 3,4seco-A -eburioadiene-4-ol 3,2'1- dioic acid 3,21-dimethyl ester (II).

Example 3.-3,4-seco-A -eburicadiene-4-ol-3,21-

dioic acid 21-methyl ester (III) A solution of 300 mg. of the dimethyl ester of 3,4- seco-A -eburica'diene-4-ol 3,2l-dioic acid (11) in 30 ml. of 6% KOH in methanol is allowed to stand at room temperature for six hours. The mixture is acidified with hydrochloric acid, the methanol removed in vacuo and the suspension extracted with chloroform. The chloroform extract is dried over sodium sulfate, filtered and the solevnt removed in vacuo. The residue (316 mg.) is recrystallized from hexane and furnishes 255 mg. of the pure 3,4-s'ec0-A -eburica'diene-4-ol-3,21- dioic acid 21-methyl ester (III) of the following properties: M.P. 148-150"; [a] +100 (c. 0.41 in CHCl Analysis0alcd. for C H O (516.74): C, 74.37; H, 10.14. Found: C, 74.33; H, 10.05.

Example 4.-3,4-seco-A -eburicene-4-ol-3,2l-dioic acid (V To a prereduced suspension of 25 mg. of 10% Pd on charcoal catalyst in alcohol (uptake 1.6- ml.) is added 25 mg. of 3,4-seco-A -eburicadiene-4-ol-3,21- dioic acid (I) in 2 ml. of alcohol. Reduction is complete after 1.4 m1. of hydrogen has been taken up (theory 1.3 mi). The solution is filtered and evaporated to dryness in vacuo. The crystalline residue upon recrystallization from methanol yields the pure 3,4-seco-A -eburicene-4-ol- 3,21-dioic acid (VI) possessing the following properties: M.P. 242-243"; [a] ,+93 (c. 0.04 in alcohol) A KB:

max.

Analysis-Coded. for C H O (504.73): C, 73.76; H, 10.38. Found: C, 73.50; H, 10.18.

Example 5.3,4-se'co-A -eburicene-4-ol-3,21-dioic acid 3,21-dimethyl ester (VII) Fifty mg. of 3,4-seco-A -eburicene-4-ol-3,2l-dioic acid (VI) is suspended in 1 ml. of methanol and methylated by treatment with ethereal 'diazomethane. The solvents are removed in vacuo yielding amorphous 3,4-seco-A eburicene-4-ol-dioic 3,21-dimethyl ester (VII).

Example 6.3,4-seco-A -eburicene-4-ol-3,2l-dioic acid 21-methyl ester (VIII) NMR (CDCl 8.75 7' (six protons, 4,4-d-imethyl).

Analysis-Calcd. for C H O (518.75): C, 74.09; H, 10.49. Found: OCH 5.98; C, 73.93; H, 10.49; OCH 6.28.

Neutralization equivalent: 513.

Example 7.-3,4-seco-A -eburicene-4-oI-3,21=dioic acid ZI-met'hyl ester (VIII) I Hydrogenation of 50 mg. of 3,4-seoo-A -eburicadiene-4-ol-3,21-dioic acid 21-methyl ester in m1. of ethan'olin the presence of 50 mg. of 10% palladium on charcoal catalyst yields 3,4-seco-A -eburi'cene-4-o1-3,21- dioic acid 21-methyl ester (VIII) after removal of the solvent in vacuo.

Example 8.-3,4-seco-A -eburicadiene-F,4,21-trio1 A' solution of 200 mg. of 3,4-seco-A -e buricadiene-4-ol-3,21-dioic acid in ml. of freshly distilled tetrahydrofuran is 'added dropwise over a fifteen minute period to a suspension of 200 mg. of lithium aluminum hydride in ml. of tetrahydrofuran. The mixture is refluxed for three hours, cooled and 025 ml. of saturated sodium sulfate solution is added. The mixture is filtered, the precipitate washed three times with hot chloroform and the solution evaporated to dryness in vacuo. The residue (197 mg.) on recrystallization from acetone gives 160 mg. of pure 3,4-'seco-A -eburicadiene-3,4,21- triol (IV) of the following properties: M.-P. 148-159"; [a] +89 ('c. 0.59 in CHCl Mrs. 3.08, 6.10, 11.28,.

Example 9.-3,4-seco-A -eburicadiene-3,4,20 -triol 3,21-diacetate (V) vTo a solution of 3,4-secoA -eburicadiene-3,4,2ltriol in anhydrous pyridine is added acetic anhydride and the mixture allowed to remain at room temperature for 18 hours. Ice is then added and the mixture further diluted with ice and water. The resulting precipitate is filtered, washed thoroughly with water, dried in vacuo and recrystallized from alcohol yielding 3,4-seco -eburicadiene-3,4,2l-triol 3,2l-diacetate (V).

Similarly, following the procedure set forth in Example 9 but substituting other acid anhydrides and acyl halides for acetic anhydride the corresponding 3,2l-diesters are formed. Thus, if benzoyl chloride or methanesulfonyl chloride or propionic anhydride are employed the respective 3,21-dibenzoate, 3,21-dimethanesulfonate and -3,21.-dipropionate of 3,4-seco-A -eburicadiene-3,4,21-

triol are produced.

Example 10.-3,4-seco-A -eburicene-4,24,28-triol- 3,21-dioic acid (IX) To a solution of 47 mg. of 3,4-seco-A -eburicadiene-4-ol-3,21-dioic acid in 3 ml. of dioxane and 0.2 ml. of pyridine is added dropwise over a thirty minute period a solution of 26 mg. of 050; in 3 ml. of dioxane. The mixture is allowed to remain at room temperature for an additional hour and then decomposed with H 5. The mixture is filtered over a Celite pad and the filtrate is evaporated to dryness in vacuo. The residue (58 mg.) is recrystallized from ethyl acetate with the aid of Darco 6-60 and furnishes the 3,4-seco-A -eburicene-4,24,28- triol-3,21-dioic acid (IX) of the following properties:

M.P. 233-235"; [Ot] j+68 (c. 0.47 in alcohol); A 2.95, 5.85;].

in ex.

Analysis.-Calcd. for C31'H52O7 (536.73): C, 69.37; H, 9.77. Found: C, 69.45; H, 9.65.

Neutralization equivalent: 269; calcd.: 268.

I CH3 H0O which comprises subjecting eburicoic acid to the action of enzymes of a microorganism of the genus Glomerella under oxidizing conditions, and recovering the product from the reaction medium.

2. The method of claim 1 wherein the microorganism is selected from the group consisting of GI merella fusarioides, Glomerella glycines and'Glomerella cingulatw.

References Cited UNITED STATES PATENTS I 3,010,878 11/1961 9 Pan l9 551 ALVIN E. TANENHOLTZ, Primary Examiner. 

